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D. Xing, X. Sun, M. Cui, J.A. Bonanno; Protection of Corneal Keratocytes by Hypoxia Preconditioning . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3845.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether hypoxia preconditioning can protect corneal keratocytes from UV–induced or epithelial scrape–induced cell death. Methods: Primary cultures of bovine corneal keratocytes were established. Second passage cells were subjected to 1.5% oxygen overnight. After 2 hours of normoxia, cells were UV–irradiated by exposure to a germicidal lamp for up to 10 minutes. Two to four hours later, cells were stained for Live–Dead or TUNEL assay. Controls were kept in normoxia for an equal period of time. For epithelial scrape–induced keratocyte death, fresh bovine eyes were hypoxia preconditioned for 6 hours, the epithelium scraped, and frozen sections were prepared for TUNEL assay four hours later. Results: Hypoxia preconditioning had its maximum protective effect on cells UV–irradiated for 8 min. Live–Dead staining showed 90% cell death in the control normoxia group and ∼1% cell death in hypoxia preconditioned cells. For TUNEL staining, hypoxia preconditioning had its maximum protective effect after 2 min. of UV–irradiation. In controls, 50.3% of cells showed apoptotic nuclei, while only 22% of cells in the hypoxia preconditioned group were TUNEL positive. In epithelial scraped corneas, 29.2% of keratocytes in the anterior third of the stroma were apoptotic in the control normoxia group and 18.8% were TUNEL positive in the hypoxia preconditioned group. The apoptotic rates in unscraped normoxic and hypoxia preconditioned corneas were 18.7% and 17.1%, respectively. Conclusions: Hypoxia preconditioning can have a protective effect on UV induced keratocyte apoptosis and epithelial scrape–induced keratocyte apoptosis. Our future studies will focus on exploring the mechanism of the hypoxic protective effect on keratocyte apoptosis.
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